In recent years it has been widely recognized that many commercial and industrial chemicals can, even in very low amounts, cause toxic effects on humans, domesticated animals, and fish and wildlife. Such toxicants can be present in trace amounts in pharmaceuticals, food additives, industrial and agricultural products and can produce acute or chronic adverse somatic effects in humans or animals exposed to or ingesting these materials, as well as mutagenic, teratogenic or carcinogenic effects. It has therefore become common place in modern society to test the newly synthesized chemical or products for an array of toxic effects. However, many of these effects are difficult to assess even though they may cause illness in humans or animals exposed to such substances. It also becomes increasingly important to be able to test processed materials or substances of unknown constituency, or food products for the presence of contaminants of a toxic character.
It is therefore quite useful to have assays which can identify toxic substances or detect them in samples of material of unknown constituency. Ideally such assays would be qualitative as well as quantitative indicating the type of chemical substance which is detected if a positive result is obtained from the assay. However the qualitative chemical analysis of an unknown sample of toxic substances is at present a very slow and expensive technological effort. Mixtures of various toxic substances can present special difficulties because of the need to conduct separate analyses for the constituents thereof. The situation can be further complicated when multiple toxic substances are present in a single sample since the interaction between the toxicants can result in additive, synergistic, or antagonistic interactions thus making the results extremely difficult to predict. Nevertheless, even if qualitative analysis is impractical, sensitive quantitative analysis of samples to be screened for the presence of deleterious, even if unknown, toxicants is of great use in determining the safety of substances in the human or animal environment.
One generalized approach to the problem of sensitive testing for the presence of adverse toxic chemicals in a sample is to use biological materials which are extremely sensitive in the assays. These bioassays typically measure the response of a biological preparation or whole organism to challenges from the test chemical or environmental sample of unknown constituents to see if the preparation or organism is affected. Such a bioassay will not identify the chemicals concerned but will quantitatively measure their effect on biological activity. It has been found that data from such bioassay tests correlate well with the effect on laboratory animals and humans when determined by conventional toxicological or epidemiological data. Various prior assays have been based on simple enzymes or group of enzyme tests or on the responses of whole organisms such as bacteria or fish which are exposed to the samples in question. One commercial system utilizes the light output of a bioluminescent bacterium to determine the biological response of the bacterium to toxicological effects of the test chemical or environmental sample being tested.
It has previously been reported by some of the inventors here that a bioassay for toxic substances is practical based on the use of submitochondrial particles. This bioassay using submitochondrial particles, known as the reverse electron transport, or RET assay has been used with another test referred to as the electron transport, ETR, to accurately predict the aquatic and cellular toxicity of a variety of chemicals. The assay is based on the use of submitochondrial particles having an intact mitochondrial membrane containing competent enzymes therein and the use of appropriate antibiotics to block the flow of electrons so that reactions can be selectively driven so as to favor reactions the product of which can be determined spectrophotometrically. A suitable reaction which may be catalyzed using the RET process is a conversion of NAD plus to NADH. The presence of toxic substances in the test sample which interfere with the functioning of competent mitochondrial enzymes or the competency of the mitochondrial lipid membrane itself would disrupt the functioning the of the RET electron flow system, the disruption of which can be detected by the change in photometric response of the solution. This bioassay is described in U.S. Pat. No. 4,808,517.
Prooxidants constitute another category of toxic substances which are capable of exerting toxicity on living organisms. Biological scientists have recently become aware that such chemicals exert their acute toxicity, mutagenicity and carcinogenicity by inducing a prooxidant state in vivo. When a prooxidant state is induced on a cellular level, the cellular concentration of activated forms of oxygen increases. The major forms of activated oxygen are the superoxide anion radical (.sup.. o.sub.2.sup.-) and its conjugate acid, the hydroperoxy radical (HO.sub.2.sup..), singlet oxygen (O.sub.2.sup.1), hydrogen peroxide (H.sub.2 O.sub.2) and the hydroxyl radical (HO.sup..). Each of these three radicals is highly reactive and has the capacity to attack cellular lipid membranes, proteins, and DNA causing their oxidative degradation. The biological consequences of the prooxidant state include inhibition of gap-junction communications between cells, sister chromatid exchanges, mutations of nuclear and mitochondrial DNA, carcinogenesis, aging, and cell death. To avoid these damaging effects, all aerobic cells have developed elaborate, multiple level defensive systems. These protective defensive systems are based on the ability of antioxidants such as vitamins A, C, and E and glutathione or enzymes such as superoxide dismutase, catalase and peroxidase to destroy the free radicals or oxidants before they can attack cellular components and exert their toxic effects.
In a normal healthy cell a delicate balance exists between activated hydrogen radicals produced as a minor by-product of aerobic respiration and other metabolic processes and the removal of these radicals by antioxidants with little oxidative damage resulting from their brief existence. However, this balance can be upset by an excessive production of free radicals or a defect in the defensive system of the cell. Exposure to such diverse chemicals as the herbicides paraquat and diquat, the anti-cancer drug adriamycin and the redox dye sulfonazo III can increase the rate of production of oxygen free radicals in vivo resulting in the creation of activated forms of oxygen faster than the cell can process them. The result is a prooxidant state which is believed responsible for the deadly lung damage seen in people exposed to paraquat, the kidney damage associated with diquat exposure, and the cardiotoxicity of adriamycin administered in cancer chemotherapy.
Because of the severe consequences of exposures to such chemicals which can induce the prooxidant state it is important that rapid and sensitive methods be developed for the detection of these substances in environmental samples or to identify new or existing chemicals with this ability. Although researchers have been able to demonstrate the ability of individual substances to generate prooxidant states using elaborate cell culture or subcellular systems, no rapid screening tests for general applicability to chemicals which induce a prooxidant state are heretofore available.